There has been great progress in our understanding of how endothelial precursor cells called angioblasts differentiate and assemble into vessels in the proper places in the embryo. In particular, the solidification of the important role of circulating endothelial precursor cells in adult neoangiogenesis has invigorated investigations into regulation of the embryonic angioblast. However, important questions regarding both differentiation and patterning of early endothelial cells remain. For example, the signals that control vascular pattern formation, the developmental stage of the cells, that respond to these signals, and how the signals are transduced by the responding cells are largely unknown. Understanding how spatio-temporal aspects of vessel formation are regulated at the molecular and cellular level is critical to our ability to reconstitute vessels in patients. In the last funding cycle we developed models to dissect vascular patterning, and we began to investigate the patterning of embryonic angioblasts. This renewal application will build upon this work and test the central hypothesis that embryonic midline signaling centers produce specific signals, both positive and negative, that act on angioblasts to pattern the vessels of the trunk. The nature of these signals will be investigated, as well as the response pathways within the target cells. We use mouse mutants in several experimental models to test hypotheses, including ES cell differentiation cultures that form primitive vessels, a novel mouse-avian embryo chimera model, and embryo explants. The specific aims are: 1) To characterize how interplay between the VEGF receptors flk-1 and fit-1 in stem-cell derived angioblasts and endothelial cells provides competence to respond to vascular patterning signals; 2) To characterize how interplay between flk-1 and fit-1 in embryonic angioblasts provides competence to respond to vascular patterning signals in vivo; 3) To determine the role of VEGF in mediating a positive patterning signal from the neural tube; and 4) To determine the role of Sonic hedgehog (Shh) in mediating a negative patterning signal from the notochord.